I am using this method to test the flavonoid content in my plant sample:
"a sample (1.0 mL) of the liquid extract was mixed with 4 mL of Milli-Q water, and 0.3 mL of aqueous NaNO2 solution (5% w/v). Subsequently, 0.3 mL of AlCl3 (10% w/v) and 2 mL of 1 M NaOH solutions were added to the mixture after 5 and 6 min, respectively. The reaction mixture was then incubated at room temperature at approximately 25 °C for 60 min. After the incubation, 200 µL of the mixture was transferred to 96-well plate in triplicate, and the absorbance was measured at 510 nm".
However, I do not understand why I need to add 4ml water, I already diluted my stock solution (50mg/ml) to different concentrations (10, 5, 2.5, and 1.25 mg/ml). I feel like this dilutes my extract even further. Is it necessary to add the water in this case?
Also, I know that Quercitin must be dissolved in methanol, but what about the other reagents (AlCl3, sodium nitrite, and sodium hydroxide) is it okay to dissolve them in water?
Thank you.
Depending on the particular flavonoid and the method used, you may make up to required volume with distilled water. Check https://www.hindawi.com/journals/tswj/2020/8780704/
Also check https://www.hindawi.com/journals/tswj/2012/923462/
Kindly look at this paper.
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0200174&type=printable
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